It has long been recognized that it is necessary to separate blood samples into their respective component parts, such as the cellular components and plasma or serum, for test purposes and other medical applications. For example, before a donor's red blood cells are tested for antigen-make up or evaluated for their compatibility with the recipient's serum prior to transfusion, they should be washed to remove proteins and resuspended in a known volume of an isotonic solution. It is well known that the separation of anticoagulated whole blood into its component parts can be effected by centrifugation whereby the red blood cells are forced under increased gravitational forces to the bottom of a centrifuged test tube thereby displacing plasma and other less dense components to higher levels. In those operations requiring merely the red blood cell layer, typically the plasma portion is decanted and the red blood cells are resuspended in a wash solution. Generally, the solution is a salt based solution. Resuspension of cells is generally accomplished by physical agitation so that any plasma which may have been trapped in intracellular spaces between the cells upon centrifugation is similarly resuspended into the solution. The solution containing the newly suspended red blood cells is then typically recentrifuged in order to repack the red blood cells together at the bottom of the tube and the supernate with the contaminating serum is again decanted. This operation of centrifugation and resuspension is generally repeated three times in order to maximize the washing of the red blood cells and remove serum which contains gamma globulin and complement components which could interfere with the Coombs Test yet still retain as many cells as possible. Although the standard recovery system results in a relatively clean population of red blood cells with minimal contamination by other elements present in the blood, there generally is a large loss of red blood cells of between 20 and 30% because of the numerous physical operations performed upon the red blood cells. Further, due to the nature of the physical operations required, automated procedures are difficult to institute and require complex, cumbersome and expensive equipment. In addition, the standard recovery systems require an average of five to six minutes per three wash cycles of the blood sample which puts an increased load upon the personnel and equipment resources when large numbers of samples must be handled.
As pointed out above, washed red blood cell suspensions are preferably used in many blood bank procedures. However, present techniques present two problems which are encountered by a technologist who wishes to prepare a suspension of washed red blood cells at some predetermined volumetric concentration. The first problem resides in the fact that the concentration of red blood cells present in various normal whole blood samples may range from 30% to 60% and may be much lower in anemic individuals. This large variation in the normal sample population prevents simple dilution from a sample of anticoagulated whole blood to prepare diluted red cells at a given predetermined concentration.
The second problem encountered by prior methods of washing red blood cells is one of time and mechanical manipulation. As pointed out above, the standard recovery systems require an average of five to six minutes per three wash cycles of the blood sample. In contrast thereto, the present method enables the red blood cells to be washed effectively and to be reconstituted to the desired percentage within less than 2 minutes.
A number of blood separation devices have been disclosed in the literature.
U.S. Pat. No. 3,932,277 to McDermott et al., directed to the separation of blood fractions, describes a system of tubes, one insertable into the other, whereby one tube inserts a barrier to separate the serum from the red blood cells after centrifuging in an attempt to prevent the mixing of the cells and the serum during decantation of the serum supernatant. During the insertion of the inner tube whereby the barrier is placed between the aforementioned portions, it is possible to have the serum filtered as it passes into the interior of the inner tube. Thus, this invention is directed towards the recovery of serum and requires great care in the placement of the barrier at the surface of the compacted red blood cell portion so as to avoid inadvertant mixing at that interface. Once in place, the barrier will prevent the removal of the red blood cells upon decantation of the serum. Thus, the barrier defeats a technician interested in working with the red blood cell layer from obtaining that cell layer. Devices, such as are disclosed in U.S. Pat. Nos. 3,799,342; 4,035,294; 4,244,694; 4,294,707; 3,687,296; 3,960,727 and 3,799,342 like the above McDermott et al. patent, are also primarily intended to facilitate recovery of the mother solution portion of the resuspension rather than the washing and suspension of the particles.
U.S. Pat. No. 3,914,985 to von Behrens is directed to a device for accurately measuring the packed mass of minor constituents of fluids, particularly whole blood, platelet-rich plasma and other body fluids. The device is particularly useful in hematology for determining the packed cell volume of platelets, lymphocytes and granularcytes. However, von Behrens makes no attempt to provide any washing and collecting of red blood cells. In fact, von Behrens states in column 7, line 29 that "unwanted cells (erythrocytes) will be disposed of entirely in the lower portion of the tube." This contrasts with one object of the present invention which is to prepare packed washed red cells and thereafter to prepare standardized concentrations thereof suspended in a compatible solution.
U.S. Pat. No. 3,075,694 to Anderson relates to a device for the separation of particles of different sizes or densities suspended in a liquid. There is, however, no disclosure therein concerning the washing of red blood cells or the reconstitution thereof in a compatible fluid.
U.S. Pat. No. 3,677,710 to Hirsch is directed to an apparatus for automatically performing the Coomb's antihuman globulin test in a continuous flow system. A techique is disclosed which permits continuous washing of the cells that are to be reacted with the Coomb's serum. This technique involves gravitational settling and separation of aggregated cells from a supernatant material. The Hirsch apparatus is quite complex and does not involve centrifugation. The present method, on the other hand, is a batch process and it is carried out in a relatively simple and inexpensive manner and yet provides fast, effective washing and resuspension of the red cells.
U.S. Pat. No. 3,858,795 to Joyce is directed to a method for washing blood cells by forcing the cells to the bottom of a stationary liquid in a container, where the liquid has a greater density at the bottom of the container than at the top. However, there is no disclosure therein of any device for preparing standarized concentrations of washed red cells.
U.S. Pat. No. 4,435,293 to Graham Jr. et al. relates to a particle washing system wherein in a preferred embodiment the fluid containing the desired particles is placed within an inner tube having near the bottom thereof an orifice with a diameter at least equal to that of the diameter of the particles, an air vent and, wherein the inner tube is positioned within an outer tube having a fluid with a density at least equal to that of the solution containing the particles to be separated within the inner tube but less than that of the particles. The application of centrifugal force to the particles directed toward the bottom of the outer tube causes the particles to move through the orifice and through the outer solution contained within the outer tube so that the particles are collected from the inner solution, washed by the outer solution and subsequently sedimented at the bottom of the outer tube. The device of Graham Jr. et al. does not disclose a method for obtaining a standarized concentration of red cells.
U.S. Pat. No. 4,436,631 to Graham Jr. et al., which patent is a continuation-in-part of the above-discussed U.S. Pat. No. 4,435,293, is similar thereto, and also does not disclose a method for obtaining a standardized concentration of red cells.
It is an object of the present invention to permit the rapid separation of particles from a solution in a "onestep" operation. It is another objective that during the separation of the particles from the solution containing the particles, the particles are washed so as to remove any non-specific serum coating and to dilute any solute drag. It is yet another objective of the present invention that these objects be accomplished in a simple system capable of economical production and employable within simple, inexpensive centrifuges commonly available. It is still yet another objective that the apparatus and methodology of the present invention be capable of replacing expensive automated cell washers presently available. Another object of the present invention is to permit a laboratory technologist to rapidly prepare standardized suspensions (with respect to concentration) of washed, red blood cells from whole blood specimens, with minimal technical manipulation. It is still another object of the present invention to provide an apparatus so that a known volume of "packed" red cells can be deposited into a receptacle, despite the fact that the concentration of red blood cells present in various normal whole blood samples may range from 30 to 60%. It is a further object of the present invention to provide a device wherein unwanted excess cells may be readily removed without disturbing desired cells.
It is yet a further object of the present invention to provide apparatus which permits facile production of a variety of cell concentrations. These any other objectives will readily become apparent to those skilled in the art in light of the teachings herein set forth.